Treatment options for patients with advanced cancer are seriously limited. Selective immune attack provides a means to combat disseminated disease. CD40-CD40L interactions have been demonstrated to be critical in the generation of optimal immune responses. We have recently demonstrated that CD40 stimulation using an agonist antibody combined with IL2 can result in highly synergistic anti-tumor responses in mice bearing advanced metastatic renal cell carcinomas (RCC) and other solid tumors. Significant protection occurred using the combination only. This protection was correlated with significant increases in dendritic cell and tumor-specific CD8+ T cell numbers and surprisingly, independent of CD4+ T cell responses. We now propose to delineate the mechanisms underlying these synergistic anti-tumor responses and optimize them. Toward this goal we have developed three specific aims. Specific Aim 1 will determine the effects of CD40 stimulation on the tumor itself. While CD40 stimulation has been shown to induce activation-induced cell death (AICD) on a variety of B cell lymphomas, the effect on solid tumors is controversial and much less clear. Preliminary data indicate that CD40 stimulation can indeed induce AICD in RCC lines and importantly, may increase susceptibility of the tumor to immune-mediated attack. Assessment of the effects of CD40 stimulation at both cellular and molecular levels will be performed. Specific Aim 2 will examine the mechanisms as to why there is no engagement of CD4+ T cells after combination treatment and how this may impair sustained anti- tumor responses. Preliminary data suggest that the CD4+ T cell undergo AICD after combination treatment due to interferon-gamma resulting in impaired secondary responses. Means to prevent this CD4+ T cell loss with subsequent effects on anti-tumor responses will be examined. Finally, Specific Aim 3 will determine the efficacy of tumor vaccines and removal of inhibitory influences such as regulatory T cells in advanced tumor-bearing mice. Preliminary data indicates that use of tumor vaccines at the time of anti-CD40 and IL-2 treatment greatly augments efficacy resulting in long-term cures. This proposal should yield significant insights, not only in the clinical extrapolation of CD40 stimulation with IL-2, but also in general immune regulation and tumor responses.